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Soybean (Glycine max) nodule bacteroids contain high concentrations of poly-,B-hydroxybutyrate and possess a depolymerase system that catalyzes the hydrolysis of the polymer.Changes in poly-p-hydroxybutyrate content Evidence for the metabolic pathway of utilization of BOHB was provided by Sierra and Gibbons (30), who demonstrated that BOHB was oxidized to acetoacetate by crude extracts from M. halodenitrificans. Acetoacetate was further metabolized when ATP, Mg2+, coenzyme A, and oxaloacetate were added to the extracts, suggesting that acetyl-CoA was an intert mediate of the reaction and that utilization occurred through the tricarboxylic acid cycle.The catalysis of nitrogen reduction by cell-free extracts of nitrogen-fixing microogranisms and nodule bacteroids requires a supply of ATP (12,23,25) and an appropriate reductant (4,8, 12,14,25). It has been estimated (3, 10) that 3 to 19 mg of carbohydrate are consumed by legume root nodules for each milligram of N2 fixed. Moreover, two or more carbon atoms are required for the export of each fixed nitrogen atom, in the forms of amino acids and amides, from the nodules to the host (33).It has been established that up to 50% of the dry weight of Rhizobium japonicum bacteroids consists of PHB (14), and that /3-hydroxybutyrate oxidation in in vitro experiments is capable of supplying the electrons for the support of bacteroid nitrogenase activity (14). The purpose of this investigation, therefore, was to study the utilization of PHB in soybean root nodules and to assess the possible role of the polymer as a source of energy for maintenance of nitrogenase activity in nodule bacteroids. MATERIALS AND METHODS CHEMICALSReagent grade chemicals or those of the highest grade available were obtained from commercial sources. NAD, sodium DL-3-hydroxybutyrate, DL-isocitrate lactone (hydrolyzed as recommended by the manufacturer), 2-mercaptoethanol, EDTA, 750 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

All species of Rhizobium except R. lupini had nitrate reductase activity. Only R. lupini was incapable of growth with nitrate as the sole source of nitrogen. However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium. Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rhizobium trifolii, R. phaseoli, and R. leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate. In bacteroids of R. japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. trifolii, R. leguminosarum, and R. phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.

The glutamine synthetase (GS) isozymes in the plant fraction of nodule extracts from 62 cultivars of Plaseolus vulgaris L. and one cultivar of Phascolus lunatus L. were analyzed by polyacrylamide gel electrophoresis. All P. vulganis nodule extracts displayed two GS activity bands: a nodule-specific band (GS.j) and a band (GS.2) similar to the single band (GSJ) present in root extracts. In nodule extracts of P. lunatus, the GS., band was detected, but the GSa band was barely detectable. In contrast to P. vulgaris, the GS.2 band and the GS, band of P. lunatus appeared to be different. The electrophoretic mobility of the GS., band in P. vulgaris was governed by both the plant cultivar and the development stage of the nodule. In nodule extracts of P. vulgaris and P. lunatus, the zone of GS., activity coincided with six to nine distinct protein bands as revealed after treatment of gels, which had previously been stained for GS activity, with Coomassie blue. All these protein bands were shown to consist of polypeptides of identical molecular weight (approximately 47,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that P. vulgaris continuously generates isozymes of GS., of increasing electrophoretic mobility during the course of nodule development.Ammonia assimilation in higher plants is considered to occur via the glutamine synthetase (GS,2 EC 6.3.1.2)/glutamate synthase (GOGAT, EC 1.4.1.14) pathway (16). Similarly, ammonia derived from nitrogen fixation by Rhizobium bacteroids in the nodules of legumes is excreted in the plant cytosol (19) where it is processed by the GS/GOGAT system (1,13,15,17,21).Since the role played by the nodule GS in the assimilation of fixed nitrogen is critical, attention has recently focused on the regulation ofthis enzyme during nodule development (4,10,20). Multiple forms of GS have been observed in various parts of plants (4,8,12,22). Two forms of GS in nodule cytosol were reported in Phaseolus vulgaris (4, 5). A nodule-specific (GS.,) form was synthesized during the late stage of nodule development. A second form GSn2), undistinguishable from the root GS (GS,), was present at all times (4, 10). In contrast, a single form ofGS was detected in nodules of all other legumes (Glycine max, Pisum sativum, Lupinus angustifolius, Cajanus cajan, Cicer arietinum, Viciafaba, Vigna unguiculata) examined (10,13,20).In 2Abbreviations: GS, glutamine synthetase; GS.,, nodule-specific glutamine synthetase; GS,2, second form of nodule GS; GSr, root GS; GOGAT, glutamate synthase. and one cultivar of P. lunatus for the presence of multiple forms of GS in the plant fraction of the nodules. We observed two forms of GS in the nodules of all cultivars of P. vulgaris and one major form and one minor form in the nodules of P. lunatus. A marked influence of plant cultivar and stage of nodule development on the electrophoretic mobility of GS., was observed in P.vulgaris. MATERIALS AND METHODS

ABSTRACTand a nonexpressing strain (Ri lupini ATCC 10318). We studied the effect of N03-on N2 fLxation activities of those legumeRhizobium combinations.The inhibitory effect of NO3-on the N2 fixation activity of legume root nodules has been under investigation for some time (14). Munns (8) and Gibson (2) have recently published thorough reviews on the subject. There are two hypotheses as to the cause of this inhibition. One has been termed the photosynthate deprivation hypothesis which attributes the decrease in N2 fixation activity to a diminished supply of photosynthate to the nodules caused by N03 reduction in the shoots (10). The other hypothesis involves a more direct effect and attributes the inhibition to the formation of N02 in the nodules by bacteroid NO3 reductase (2). Nitrate itself seems not to affect N2 fixation activity in cultures of Rhizobium sp. 32HI (I 1), but N02 inhibits fixation in cultures of Rhizobium sp. 32H1 (11), Rhizobium japonicum bacteroid suspensions (12), and crude R japonicum bacteroid nitrogenase extracts (5). In addition, N02 may form a NO compound with leghemoglobin (13) and, thus, prevent leghemoglobin from binding 02, which could interfere with the N2-fixing process.The presence of an active N03 reductase in some Rhizobium bacteroids has been well documented (1,(5)(6)(7) To determine N02 content of nodules, 0.5 g of nodules was macerated in 1 ml of 1 M zinc acetate with a mortar and pestle. After maceration, the total volume was adjusted to 5 ml with deionized H20, and cellular debris was removed by centrifugation at 12,000g for 10 min. One ml of the supernatant was combined with 1.7 ml of 1 M zinc acetate and 1 ml 95% ethanol and centrifuged at 3,000g for 20 min to remove precipitated proteins.The supernatant from the second centrifugation was assayed quantitatively for N02-by a colorimetric method (9). Nitrite content was expressed as nmol NO2J/g nodule fresh weight. Each